Page 72 - 77_01

FIBROBLAST
GROWTH
FACTOR
RECEPTOR
3….

4. DISCUSSION

RNA
interference
is
emerging
as
a
strategy
for
the
highly
specific

suppression
of
gene
expression,
both
in
vitro
and
in
vivo.
The
present
work
shows

the
use
of
siRNA
duplexes
targeting
FGFR3
to
knockdown
the
mRNA
and
protein

expression
levels
of
this
receptor
in
immortalized
human
chondrocytes
carrying

the
achondroplasia
mutation
(G380R).

Our
results
are
consistent
with
previous
studies
demonstrating
that
siRNAs

targeting
distinct
mRNA
sequences
results
in
different
silencing
efficiency
(11,
13).

A
significant
decrease
of
FGFR3
mRNA
and
protein
levels
was
observed
after

transient
transfection
with
siRNA
sequences
1
and
2,
while
the
siRNA
sequence
3

did
not
induce
FGFR3
down-­-regulation
in
the
same
extent.

Activation
of
FGFR3
results
in
trans-­-autophosphorylation
of
juxtaposed

intracellular
kinase
domains
with
subsequent
recruitments
of
signalling
molecules

leading
to
phosphorylation
and
activation
of
different
signalling
pathways.

Therefore,
we
determined
whether
these
signalling
pathways
were
also
affected

by
FGFR3
knockdown.
In
particular,
we
have
analyzed
one
of
the
best
known
and

characterized
downstream
signal
transduction
pathways
coupled
to
FGFR3,
the

ERK
mitogen
activated
protein
kinase
cascade.
The
sustained
activation
of
this

pathway
is
involved
in
the
inhibitory
effect
of
FGF
signalling
on
chondrocyte

proliferation
and
cartilage
matrix
production
(3,
5).
FGFR3
knockdown
by
siRNAs

(siRNAs
sequence
1
and
2)
attenuated
the
ERK1/2
phosphorylation
suggesting

that
the
negative
effects
mediated
by
this
pathway
could
be
counteracted
by

siRNAs
targeting
FGFR3.

RNA
interference
strategies
have
been
previously
described
in
different

types
of
cancer
cells
that
have
activating
mutations
in
FGFR3.
In
bladder
cancer

cells,
knockdown
of
FGFR3
by
siRNAs
lead
to
decrease
proliferation,
reduced

clonogenicity
and
soft
agar
growth
(14).
Similarly,
siRNA-­-mediated
knockdown
of

FGFR3
inhibited
anchorage-­-independent
growth
of
adrenal
carcinoma
cells
(12).

Inhibition
of
FGFR3
expression
by
RNA
interference
has
been
also
reported
in

multiple
myeloma
cells
(11).
On
the
other
hand,
regarding
skull
and
skeletal

growth
disorders,
RNA
interference
to
knock-­-down
the
expression
of
a
mutant

FGFR2
has
been
used
in
Apert
syndrome,
a
classic
severe
form
of
craniosynostosis

(15).
However,
to
our
knowledge
we
have
shown
for
the
first
time
the
use
of
RNA

interference
targeting
FGFR3
in
human
achondroplastic
chondrocytes.

Although
several
therapeutic
approaches
have
emerged
for
achondroplasia

treatment,
the
translation
of
these
therapies
into
the
clinic
has
not
taken
place.
One

of
the
main
troubles
for
the
success
of
new
therapies
is
the
delivery
of
compounds

to
growth
plate
chondrocytes
of
cartilage.
The
avascular
nature
of
the
cartilage

tissue
represents
a
challenge
for
drug
delivery.
Conjugation
of
siRNAs
with
small

molecules
that
posses
affinity
for
cartilage
or
chondrocytes
could
facilitate
the

target
to
growth
plate
chondrocytes
of
cartilage.
In
this
sense,
several
strategies

have
been
developed
to
promote
cell/tissue-­-specific
siRNA
delivery
(16).

Additionally,
the
dense
extracellular
matrix
that
surround
chondrocytes
form
an

important
barrier
for
drug
delivery.
However,
siRNAs
are
small
enough
that

diffusion
through
extracellular
matrix
should
not
be
an
issue.

9